4.11. Generation of FAH-G337S Mutant HEK Cells by CRISPR/Cas9-Mediated Genomic Edition

The FAH missense mutation (c.1009G > A) was introduced by targeted homologous recombination at the endogenous FAH locus in human embryonic kidney (HEK) cells. HEK293 cells were co-transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham MA) with the p.X459-FAH -sgRNA plasmid (2.5 µg/well) and single strand oligonucleotide (ssODN) (7.4 µg) containing the FAH-G337S mutation as well as flanking sequences on each side of the targeted exon. The pX459 plasmid was obtained from Addgene (Ref. 62988) and expresses Cas9 from S. pyogenes, puromycin resistance gene and cloning backbone for sgRNA. The targeted sequence was designed next to a NGG sequence named protospacer adjacent motif (PAM, N can be any nucleotide). Additionally, a silent substitution was introduced in the PAM sequence of the ssODN in order to prevent secondary cleavage after homology directed repair (HDR) at the FAH locus and enhance recombination efficiency. Transfected cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C and 5% CO₂ for 22 h, after which media were replaced and RS-1 reagent (Sigma, Saint Louis, MO, USA) was added at 10 µM final concentration in order to stimulate HDR. Cells were incubated under the same conditions for another 5 h and then puromycin was added to media in a final concentration of 1 µg mL⁻¹, in order to select only the transfected clones. Cells were maintained under these conditions for another 2 days, followed by a limiting dilution sub-cloning assay on 96-well plates. Completely isolated cells were progressively grown and passed to bigger dishes in order to obtain monoclonal cell lines. Genomic DNA was extracted from each of these clones and targeted exons were PCR-amplified for FAH gene sequencing to characterize genome editing at the molecular level. As HEK cells are triploid on average, the sequencing analysis is consistent with two alleles resulting from HDR modification were detected in each HEK293-FAHG337S cellular clone, while the third one remained wild type. Successfully modified clones were selected for their phenotypic characterization. Consistently, FAH-targeted clones exhibited accumulation of succynilacetone and hydroxyphenylpyruvic acid, as determined by NMR spectroscopy.